How RhoGDI binds Rho

Like all Rho (Ras homology) GTPases, RhoA functions as a molecular switch in cell signaling, alternating between GTP- and GDP-bound states, with its biologically inactive GDP-bound form maintained as a cytosolic complex with RhoGDI (guanine nucleotide-exchange inhibitor). The crystal structures of RhoA–GDP and of the C-terminal immuno­globulin-like domain of RhoGDI (residues 67–203) are known, but the mechanism by which the two proteins interact is not known. The functional human RhoA–RhoGDI complex has been expressed in yeast and crystallized (P6₅22, unit-cell parameters a = b = 139, c = 253 Å, two complexes in the asymmetric unit). Although diffraction from these crystals extends to 3.5 Å and is highly anisotropic, the experimentally phased (MAD plus MIR) electron-density map was adequate to reveal the mutual disposition of the two molecules. The result was validated by molecular-replacement calculations when data were corrected for anisotropy. Furthermore, the N-­terminus of RhoGDI (the region involved in inhibition of nucleotide exchange) can be identified in the electron-density map: it is bound to the switch I and switch II regions of RhoA, occluding an epitope which binds Dbl-like nucleotide-exchange factors. The entrance of the hydrophobic pocket of RhoGDI is 25 Å from the last residue in the RhoA model, with its C-terminus oriented to accommodate the geranylgeranyl group without conformational change in RhoA.